Screening Methods

ABSTRACT

The present invention relates a mammalian polypeptide designated Inhibitory PAS Domain Protein (IPAS) which polypeptide is useful for the inhibition of angiogenesis and/or tumor progression. The invention also relates to screening methods for compounds potentially useful as medicaments for the treatment of medical conditions related to angiogenesis or tumor progression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from Swedish Patent Application No.0002551-0, filed Jul. 6, 2000, and U.S. Provisional Patent ApplicationSer. No. 60/217,570, filed Jul. 12, 2000. These applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a mammalian polypeptide designatedInhibitory PAS Domain Protein (IPAS) which polypeptide is useful for theinhibition of angiogenesis and/or tumor progression. The invention alsorelates to screening methods for compounds potentially useful asmedicaments for the treatment of medical conditions related toangiogenesis or tumor progression.

BACKGROUND ART

Oxygen plays a critical biological role as the terminal electronacceptor in the mitochondria of vertebrate cells. During evolution,these cells have developed ways to sense alterations in oxygen levelsand, during this process, acquired the ability to conditionally modulatethe expression of genes involved in adaptive physiological responses tohypoxia including angiogenesis, erythropoiesis, and glycolysis. Thesegenes include vascular endothelial growth factor, eryhtropoietin,several glycolytic enzymes and inducible nitric oxide synthase, and haveall been shown to contain hypoxia responsive elements (HREs) (forreviews, see Guillemin and Krasnow (1997) Cell 89, 9–12; Wenger andGassmann (1997) Biol. Chem. 378, 609–616). Under hypoxic conditionsthese response elements are recognized by a heterodimeric complexconsisting of the hypoxia inducible factor-1α (HIF-1α) and Arnt (Wang etal. (1995) Proc. Natl. Acad. Sci. USA 92, 5510–5514; Gradin et al.(1996) Mol. Cell. Biol. 16, 5221–5231). Both these transcription factorsbelong to the rapidly growing family of basic-helix-loop-helix(bHLH)-PAS (Per, Arnt, Sim) proteins.

A family of helix-loop-helix proteins designated Id¹⁸ has beenidentified as antagonists of bHLH transcriptional regulators. bHLHproteins typically bind regulatory sequences in a heterodimericconfiguration and function to activate differentiation-linked geneexpression. The heterodimer usually comprises a class A bHLH proteintogether with a class B bHLH protein. In the presence of excess Idprotein, the class A bHLH partner is typically titrated out throughheterodimerization with Id protein.

Dysregulation or overfunction of HIF-1α might cause a variety ofpathological conditions including tumor progression¹⁵ ¹⁶ ⁷ andinflammatory angiogenesis¹⁷. Consequently, there is a need foridentification of compounds acting as negative regulators of HIF-1α,said compounds being potentially useful against medical conditionsrelated to angiogenesis and tumor progression.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Mouse IPAS sequence and expression

(a)

Deduced amino acid sequence of mouse IPAS (SEQ ID NO:3). Domains ofbasic helix-loop-helix and PAS A and B are shown.

(b)

Schematic representation of structural features of IPAS and thehypoxia-inducible factors. The percent identities of each protein withIPAS within the bHLH and PAS motifs are shown. bHLH, basichelix-loop-helix; PAS, Per/Arnt/Sim; N- or C-TAD, N- or C-terminaltransactivation domain.

(c)

Northern blot analysis of IPAS expression in adult mouse tissues.Poly(A)⁺ RNA(4.5 μg) from various adult mouse tissues were hybridizedwith ³²P-labeled IPAS cDNA probe. The positions of RNA markers are shownon the right in kb.

(d)-(o)

In situ hybridization analysis. Sections of cornea (d-g), retina (h-k),and cerebellum (l-o) of adult mouse were hybridized with antisense RNAporbes of mIPAS (d, e, h, i, l, and m) or mHIF-1α (f, g, j, k, n, ando). Light—(d, f, h, j, l and n) and dark-field (e, g, i, k, m, and o)views are shown. C, cornea; Ep, epithelium; S, substantia propria; LE,lens epithelium; GC, granular cell layer; INL, inner nuclear layer; R&C,rods and cones, G, granular layer; P, Purkinje cells; M, molecularlayer.

FIG. 2: IPAS is a dominant negative regulator of hypoxia-induciblefactors

(a)

IPAS does not transactivate HRE-driven reporter gene. Increasing amountsof IPAS expression vector (CMV IPAS) were cotransfected withHRE-luciferase reporter gene into HeLa cells. The cells were culturedunder either normoxic (21% O₂) or hypoxic (1% O₂) conditions for 24 hand the cellular luciferase expression was determined.

(b), (c)

IPAS inhibits hypoxia-inducible factors-mediated gene expression. IPASexpression vector, HRE-luciferase reporter, and HIF-1α (b) or HLF (c)expression vector (CMV HIF-1α or CMV HLF, respectively) were introducedinto HeLa cells. After 24 h-incubation in normoxic (21% O₂) or hypoxic(1% O₂) condition, luciferase activity was measured. Results wereexpressed as fold induction compared with the luciferase activity in thecells transfected with reporter gene alone. Means±SD were shown.

(d)

IPAS does not affect on HIF-1α and HLF protein levels. HeLa cells weretransfected with FLAG-tagged IPAS expression plasmid (1.0 μg/21.5 cm²dish) and exposed to hypoxia for 6 h. Whole cell extracts (50 μg) wereprepared and subject to immunoblot analysis using antibodies againstHIF-1α (Novus), HLF (Novus), and FLAG epitope (Sigma) essentially aspreviously described ⁶.

FIG. 3: IPAS specifically attenuates hypoxia-inducible mRNAs expression

(a)

Hypoxia-inducible gene expression is impaired in IPAS overexpressingcells. Wild type(Hepa1c1c7) or IPAS-stably transfected (Hepa IPAS) mousehepatoma cell lines were cultured in either normoxic (N) or hypoxic (H)conditions for 24 h. Poly(A)⁺ RNA from the cells were separated andhybridized with radiolabeled mouse IPAS, PGK1, VEGF, and β-actin cDNAprobe as indicated.

(b)

Inhibition of hypoxia-inducible gene expression by IPAS attranscriptional level. HRE-luciferase reporter was transfected with orwithout HIF-1α expression vector into Hepa1c1c7 or Hepa IPAS cells. Thecells were cultured under conditions of either 21% or 1% O₂concentration and subject to luciferase assay. Luciferase content of theHepa1c1c7 cells transfected with the reporter gene alone was served as acontrol and results were shown as fold induction compared with thecontrol.

(c)

IPAS inhibits the binding of HIF-1α/Arnt complex to HRE. Nuclearextracts from normoxic or hypoxic Hepa1c1c7 and Hepa IPAS cells wereanalyzed by EMSA using ³²P-labeled HRE oligonucleotide probe. Theasterisks shows position of constitutive HRE-binding activity in thenuclear extracts and arrow shows the position of hypoxia-inducedHIF-1α/Arnt-DNA complex ¹². Competition assay by unlabeled HRE (S) orunrelated sequence(NS) and supershift formation by anti HIF-1α antibodyand anti-Arnt antibody were shown.

(d)

IPAS does not affect on dioxin-inducible gene expression. Hepa1c1c7 andHepa IPAS cells were treated with or without TCDD (10 nM) for 24 h andpoly(A)⁺ RNA from the cells was hybridized with ³²P-labeled mouseCYP1A1, IPAS, and β-actin cDNA probe.

(e)

IPAS has no effect on TCDD-mediated XRE-reporter gene expression.Hepa1c1c7 and Hepa IPAS cells were transfected with XRE-reporter plasmidand after incubation with or with out TCDD for 24 h luciferase activitywas monitored. Results were expressed as fold induction of luciferaseactivity compared to the ligand free control of Hepa1c1c7 cells.Means±SD were shown.

FIG. 4: IPAS targets HIF-1α to form a nonfunctional complex

(a)

IPAS physically interacts with HIF-1α. In vitro-translated GST-IPAS orGST was mixed with ³⁵S-labeled, in vitro translated Arnt or HIF-1α andimmunoprecipitation with anti-GST antibody was carried out. Theprecipitant was separated by SDS-PAGE followed by autoradiography. Forthe loading control, 10% of input Arnt and HIF-1α were shown.

(b)

The N-terminal structure of HIF-1α is essential for theheterodimerization with IPAS. ³⁵S-labeled, in vitro translated IPAS wasincubated with GAL4-fusion of various fragments of HIF-1α and subject tothe immunoprecipitation with either anti GAL4 antibody or preimmunecontrol serum. Precipitated fraction was analyzed by SDS PAGE andresults were obtained by autoradiography. Ten percent of input IPAS wasshown as a control.

(c),(d)

In vivo interaction between IPAS and HIF-1α. COS7 cells were transfectedwith various amounts of expression vectors for GAL4-HIF-1α/1-330 andVP16-IPAS (c) or GAL4-IPAS and VP16-Arnt (d) as indicated together withGAL4-driven reporter gene. After 24 h-incubation, cellular luciferaseactivity was determined. Results were expressed as fold inductioncompared with the luciferase contents of the cells transfected withreporter gene alone.

(e)

IPAS/HIF-1α heterodimer fails to bind to HRE. Various combinations of invitro translated IPAS, HIF-1α, and Arnt, or unprogrammed reticulocytelysate as indicated were mixed with ³²P-labeled HRE oligonucleotideprobe, and the protein-DNA complex formation was monitored by EMSA.Results were visualized by autoradiography.

FIG. 5: Involvement of IPAS in silencing the production of angiogenicgrowth factor in cornea epithelium cells

Primary culture of mouse cornea epithelium cells were transfected witheither antisense IPAS expression plasmid or empty vector (vector) andincubated under normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditionsfor 24 h. Total RNAs from the cells were extracted and Northern blotanalysis using radiolabeled mouse VEGF cDNA probe was performed. TotalRNAs form normoxic and hypoxic Hepa1c1c7 cells were supplied as areference for VEGF induction. As a loading control, 18S RNA levels areshown.

DISCLOSURE OF THE INVENTION

The present invention provides a model wherein activated HIF-1αencounters a negative regulation by a small protein factor such as IPAS,to form a nonfunctional heterodimeric complex. This mode of regulationof HIF-1α might contribute to a fine-tuning of hypoxia signaling in situas evidenced by profound negative effect of IPAS in corneal VEGFproduction. On the other hand, ectopic expression of IPAS potentiallyrepressed hypoxia-inducible VEGF expression, and the negative effect ofIPAS was selective to hypoxia signaling so far tested. Therefore, it ispostulated that IPAS is useful as a target in therapeutic drug designfor various angiogenic diseases, such as ischemic cardiovascularlesions, stroke, and diabetic microvascular diseases.

Consequently, in a first aspect this invention provides an isolatednucleic acid molecule selected from:

(a) nucleic acid molecules comprising a nucleotide sequence set forth asSEQ ID NO: 2;

(b) nucleic acid molecules comprising a nucleotide sequence capable ofhybridizing, under stringent hybridization conditions, to a nucleotidesequence complementary to the polypeptide coding region of a nucleicacid molecule as defined in (a) and which codes for a biologicallyactive mammalian lIPAS polypeptide or a functionally equivalent modifiedform thereof; and

(c) nucleic acid molecules comprising a nucleic acid sequence which isdegenerate as a result of the genetic code to a nucleotide sequence asdefined in (a) or (b) and which codes for a biologically activemammalian WAS polypeptide or a functionally equivalent modified formthereof.

The nucleic acid molecules according to the present invention includescDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, andcombinations thereof. Genomic DNA may be obtained by screening a genomiclibrary with the IPAS cDNA described herein, using methods that are wellknown in the art. RNA transcribed from DNA is also encompassed by thepresent invention.

The term “stringent hybridization conditions” is known in the art fromstandard protocols (e.g. Ausubel et al., supra) and could be understoodas e.g. hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1%SDS at +68° C.

In a prefeffed form of the invention, the said nucleic acid molecule hasa nucleotide sequence identical with SEQ ID NO: 2 of the SequenceListing. However, the nucleic acid molecule according to the inventionis not to be limited strictly to the sequence shown as SEQ ID NO: 2.Rather the invention encompasses nucleic acid molecules carryingmodifications like substitutions, small deletions, insertions orinversions, which nevertheless encode proteins having substantially thebiochemical activity of the IPAS polypeptide according to the invention.Included in the invention are consequently nucleic acid molecules, thenucleotide sequence of which is at least 90% homologous. preferably atleast 95% homologous, with the nucleotide sequence shown as SEQ ID NO: 2in the Sequence Listing.

Included in the invention is also a nucleic acid molecule whichnucleotide sequence is degenerate, because of the genetic code, to thenucleotide sequence shown as SEQ ID NO:2. A sequential grouping of threenucleotides, a “codon”, codes for one amino acid. Since there are 64possible codons, but only 20 natural amino acids, most amino acids arecoded for by more than one codon. This natural “degeneracy”, or“redundancy”, of the genetic code is well known in the art. It will thusbe appreciated that the nucleotide sequence shown in the SequenceListing is only an example within a large but definite group ofsequences which will encode the IPAS polypeptide.

In a further aspect, this invention provides an isolated mammalian IPASpolypeptide encoded by the nucleic acid molecule as defined above. In apreferred form, the said polypeptide has an amino acid sequenceaccording to SEQ ID NO: 3 of the Sequence Listing. However, thepolypeptide according to the invention is not to be limited to apolypeptide with an amino acid sequence identical to SEQ ID NO: 3 in theSequence Listing. Rather the invention encompasses polypeptides carryingmodifications like substitutions, small deletions, insertions orinversions, which polypeptides nevertheless have substantially thebiological activities of the IPAS polypeptide. Included in the inventionare polypeptides, the amino acid sequence of which is at least 90%homologous, preferably at least 95% homologous to the amino acidsequence shown as SEQ ID NO: 3 in the Sequence Listing.

In yet another aspect, the invention provides a vector comprising thenucleic acid sequence as defined above The term “vector” refers to anycarrier of exogenous DNA that is useful for transferring the DNA to ahost cell for replication and/or appropriate expression of the exogenousDNA by the host cell. The said vector can be a replicable expressionvector, which carries and is capable of mediating the expression of anucleic acid sequence according to the invention. In the presentcontext, the term “replicable” means that the vector is able toreplicate in a given type of host cell into which it has beenintroduced. Examples of vectors are viruses such as bacteriophages,cosmids, plasmids and other recombination vectors. Nucleic acidmolecules are inserted into vector genomes by methods well known in theart.

Included in the invention is also a cultured host cell harboring avector according to the invention. Such a host cell can be a prokaryoticcell, a unicellular eukaryotic cell or a cell derived from amulticellular organism. The host cell can thus e.g. be a bacterial cellsuch as an E. coil, cell; a cell from a yeast such as Saccharomycescervisiae or Pichia pastoris, or a mammalian cell. The methods employedto effect introduction of the vector into the host cell are standardmethods well known to a person familiar with recombinant DNA methods.The invention also includes a process for production of a mammalian IPASpolypeptide, comprising culturing the said host cell under conditionswhereby said polypeptide is produced, and recovering said polypeptide,

In a further important aspect, the invention provides a (screening)method for identifying an agent useful for activating the expression ofa mammalian IPAS nucleic acid molecule, said method comprising the steps

(i) contacting a candidate agent with a mammalian IPAS nucleotide acidmolecule, or with a mammalian IPAS polypeptide, according to theinvention; and

(ii) determining whether said candidate agent activates the expressionof the said mammalian IPAS nucleic acid molecule, or stimulates thebiological activities of the said polypeptide.

For screening purposes, appropriate host cells can be transformed with avector having a reporter gene under the control of the IPAS geneaccording to this invention. The expression of the reporter gene can bemeasured in the presence or absence of an agent with known activity(i.e. a standard agent) or putative activity (i.e. a “test agent” or“candidate agent”). A change in the level of expression of the reportergene in the presence of the test agent is compared with that effected bythe standard agent. In this way, active agents are identified and theirrelative potency in this assay determined.

As used herein, the term “reporter gene” means a gene encoding a geneproduct that can be identified using simple, inexpensive methods orreagents and that can be operably linked to an IPAS sequence. Reportergenes such as, for example, a luciferase, β-galactosidase, alkalinephosphatase, or green fluorescent protein reporter gene, can be used todetermine transcriptional activity in screening assays according to theinvention (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185,San Diego: Academic Press, Inc. (1990); see also Sambrook, supra).

As used herein, the term “agent” means a biological or chemical compoundsuch as a simple or complex organic molecule, a peptide, a protein or anoligonucleotide. Such an agent, identified in the methods according tothe invention, is potentially useful e.g. in the identification,development and manufacture of medicaments for the inhibition ofangiogenesis and/or tumor growth, including angiogenic diseases relatedto ischemic cardiovascular lesions, stroke, or diabetic microvasculardiseases.

Consequently, the invention also provides a method for the treatment ofangiogenic disease or tumor growth, comprising administering to asubject an effective amount of an agent identified by the methoddescribed above. The term “treatment” means any treatment of a diseasesin a mammal, including: (i) preventing the disease, i.e. causing theclinical symptoms of the disease not to develop; (ii) inhibiting thedisease, i.e. arresting the development of clinical symptoms; and/or(iii) relieving the disease, i.e. causing the regression of clinicalsymptoms. The term “effective amount” means a dosage sufficient toprovide treatment for the disease state being treated. This will varydepending on the patient, the disease and the treatment being effected.

Experimental Methods

Throughout this description the terms “standard protocols” and “standardprocedures”, when used in the context of molecular biology techniques,are to be understood as protocols and procedures found in an ordinarylaboratory manual such as: Current Protocols in Molecular Biology,editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook,J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A laboratorymanual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 1989.

Plasmid Construction

pcDNA3 IPAS was made by insertion of the EcoRI-NotI fragment from pT7T3DIPAS (GenBank Acc: AA028416) into EcoRI-NotI digested pcDNA3 plasmid(Invitrogen). PCMV IPAS or pFLAG IPAS contained HindIII-XbaI orBamHI-XbaI fragment from pcDNA3 IPAS in corresponding site of pCMV4 orpCMV FLAG plasmid, respectively⁶. HRE-luciferase, XRE-luciferase, andpCMV HIF-1α are described elsewhere¹² ²⁰. pBluescript mHLF was gift fromDr. Y. Fujii-Kuriyama and used for construction of pCMV mHLF. For theconstruction of the pGST, a plasmid for in vitro translation ofGST-fusion protein, GST cDNA and multiple cloning site of pGEX-4T-3(Amersham Pharmacia Biotech) was amplified by PCR with BglII andHindiIII linker, and subcloned into BglII-HindIII site of pSP72 vector(Promega). pGST IPAS was made by insertion of PCR-cloned IPAS cDNA withBamHI and XhoI linker into BamHI-XhoI-digested pGST. GAL4 HIF-1α/1-826,1-330, 1-652, 526-826 were as previously described⁶. For pCMX GAL4-IPASor pCMX VP-16 IPAS construction, EcoRI-XbaI or BamHI-XbaI fragment ofpcDNA3 IPAS was inserted into EcoRI-NheI site of pCMX GAL4 or toBamHI-NheI site of pCMX VP16, respectively. PCMXVP16-Arnt was gift fromDr. I. Pongratz. To make antisense IPAS expression plasmid, full lengthIPAS cDNA with EcoRI-BamHI linker was inserted in inverted direction toBamHI-EcoRI site of pcDNA3 plasmid.

Cell Culture and Transfection

Hepa1c1c7, HeLa, and COS7 cells were from ATCC. Hepa IPAS cells wereestablished by stable transfection of Hepa1c1c7 cells with pEFIRESpuroIPAS and puromycin (5 μg/ml) selection. Transient transfections werecarried out by the lipofection procedure in 28 cm² culture plates. Inluciferase assay, 0.5 μg of reporter plasmids and indicated amountsexpression plasmids were transfected. Hypoxic- or TCDD treatment of thecells was previously described¹².

Northern Blot and In Situ Hybridization Analysis

Poly(A)⁺ RNAs (4.5 μg) from various tissues of 8 week-old C57B16 mice orHepa1c1c7 and Hepa IPAS cells were obtained by guanidiumthiocyanatemethods followed by oligo dT-beads purification (Dynal) and analyzed byNorthern blot using ³²P-labeled cDNA probes of mIPAS (nt 623–897), mPGK1(nt 426–771), mVEGF3 (nt 24–466), mCYP1A1 (nt 874–1199), and β-actin (nt930–1075). Total RNA (20 μg) from mouse corneal epithelium primaryculture or Hepa1c1c7 cells was separated and probed with radiolabeledmVEGF3 cDNA (nt 24–466) probe. In situ hybridization of tissue sectionsfrom 8 week-old C57B16 mice using ³⁵S-labeled mIPAS or mHIF-1α antisenseRNA probe was performed as previously described²¹.

Electrophoretic Mobility Shift Assay

Nuclear extracts from either normoxic or hypoxic cells were prepared asdescribed before¹². Ten microgram of the nuclear extract was incubatedwith ³²P-labeled HRE oligonucleotide in a buffer containing 0.1 μg ofsonicated, denatured calf thymus DNA in 10 mM Tris-HCl (pH 7.5), 60 mMKCl, 50 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 5 mM dithiothreitol (DTT), 5%glycerol. Various combination of in vitro translated proteins (5 μleach) were mixed with HRE probe in a solution containing 10 mM Hepes,100 mM KCL, 0,1 mM EDTA, 3 mM MgCl₂, 4 mM spermidine, 0.5 mM DTT, 10%glycerol, 20 ng/μl tRNA, 1 ng/μl salmon-sperm DNA. The protein-DNAcomplexes were separated on 4% polyacrylamid gel in 0.5× TBE buffer(1×TBE; 89 mM Tris, 89 mM Boric acid, 5 mM EDTA).

In Vitro Protein Interaction Assay

GST-fused IPAS or GAL4-fusion of various fragments of HIF-1α weregenerated by translation either in the presence or absence of³⁵S-labeled methionine in rabbit reticulocyte lysate (Promega). Proteinconcentration of GST-IPAS or GAL4- HIF-1αs was determined on the basisof incorporated ³⁵S-labeled methionine. Equal amount of ³⁵S-labeled, invitro translated Arnt, HIF-1α or IPAS were incubated with GST IPAS orGAL4- HIF-1αs for 1 h at room temperature, followed by incubation withanti-GST antibody (Amersham Pharmacia Biotech) or anti-GAL4 antibody(Upstate Biotechnology) conjugated Protein A Sepharose (AmershamPharmacia biotech) for another 1 h at room temperature. After briefcentrifugation, coimmunoprecipitated proteins were analyzed by SDS-PAGE.

Isolation of Murine Corneal Epithelial Cells

Six-week-old C57B16/J healthy mice were killed with a lethal dose ofCO₂. The eyes were enucleated and the corneal tissue was dissected inDME medium supplemented with 10% bovine calf serum under astereomicroscope. The corneal tissue was cut into small pieces understerile conditions and washed with DMEM twice. The tissue masses wereplaced onto a gelatin-coated tissue culture plate and incubated in DMEMwith 10% bovine calf serum supplemented with human recombinant FGF-β atthe concentration of 3 ng/ml. After incubation in 5% CO₂ for 8 days,corneal epithelial cells grown to nearly confluence were trypsinized. Asingle cell suspension was then seeded onto 21.5 cm² culture dishes andcells were grown under the same condition as described above.

EXAMPLES Example 1 Identification of IPAS Sequence

Hidden Markov profiles³ were designed using the HMMER 1,8,3 software⁹from nucleotide sequences corresponding to the PAS domain of a selectednumber of bHLH/PAS factors. A mouse EST database at GenBank was screenedand an EST clone of 460 bp (GenBank Acc: AA028416; SEQ ID NO:1)containing a bHLH (basic-helix-loop-helix) PAS motif, was identified.

DNA sequence analysis revealed that IPAS cDNA (SEQ ID NO: 2) contains anopen reading frame of 921 nucleotides, encoding a polypeptide of 307amino acids (FIG. 1 a; SEQ ID NO: 3). The predicted polypeptide wasdesignated IPAS (Inhibitory PAS Domain Protein)

Alignment analysis of this amino acid sequence with known bHLH PASfactors showed high similarity to HIF-1α⁴ and HLF⁵ in the N-terminalbHLH domain (75% and 76% identity, respectively; FIG. 1 b), and to alesser extent within PAS region (34% and 36% in the PAS A, and 40% and36% in the PAS B domain, respectively; FIG. 1 b). Notably, IPAS lacksthe sequence corresponding to C-terminal region of HIF-1α and HLF, inwhich two transactivation domains (NTAD and CTAD) have been identified.

Example 2 IPAS mRNA is Expressed Predominantly in the Eye

Northern blot analysis of poly(A)⁺ RNA from a variety of mouse tissuesdemonstrated that IPAS mRNA was expressed predominantly in the eye andat lower levels in the cerebellum and the cerebrum. No obviousexpression in was detected in other tested mouse tissues, indicating avery tissue-restricted expression pattern of IPAS mRNA (FIG. 1 c).

Example 3 IPAS Expression is Observed in the Epithelial Cell Layer ofthe Cornea

To characterize the spatial expression pattern of the IPAS gene in theeye and cerebellum, in situ hybridization was performed. Intense IPASexpression was observed in the epithelial cell layer of the cornea (FIG.1 d and 1 e) and with lower intensity in the layers of ganglion cells,inner nuclear cells, and rods and cones of the retina (FIG. 1 h and 1i).

Expression of HIF-1α mRNA was detected by in situ hybridization at lowlevels in the epithelium of the cornea (FIG. 1 f and 1 g), demonstratingremarkably dominant expression of IPAS over HIF-1α in these cells.HIF-1α was also expressed in the same layers of retina where IPASexpression was observed (FIG. 1 j and 1 k), In the cerebellum,expression of IPAS was limited to the Purkinje cell layer (FIG. 1 l and1 m), whereas HIF-1 α did not show any localized expression throughoutthe sections (FIG. 1 j and 1 k). Both IPAS and HIF-1α mRNAs wereobserved as weak diffuse signal over nonspecific background levels incertain areas of the cerebrum (data not shown).

Example 4 Coexpression of IPAS Reduces Hypoxia-Inducible Reporter GeneExpression in HeLa cells

The structural similarity of IPAS to hypoxia-inducible transcriptionfactors and the colocalization of IPAS and HIF-1α in mouse corneaprompted us to investigate the role of IPAS in transcriptional controlof cellular responsiveness to hypoxia. We performed in HeLa cells atransient transfection assay using a hypoxia-response element- (HRE-)driven luciferase reporter in the absence or presence of transientlyexpressed IPAS. Incubation of the cells under hypoxic (1% O₂) conditionsinduced 4.2-fold activation of the reporter gene, representing theinduced transactivation function of endogenous hypoxia-inducible factors(FIG. 2 a). Coexpression of IPAS reduced hypoxia-inducible reporter genein HeLa cells stimulated to a high level of luciferase expression inhypoxia-dependent manner (FIG. 2 b and FIG. 2 c, respectively),indicating that IPAS acts as a dominant negative regulator of thefunction of endogenous hypoxia-inducible factors. IPAS had no effect onhypoxia-induced protein stabilization of HIF-1α and HLF (FIG. 2 d),which has previously been shown to represent a critical initial step inthe activation of HIF-1α or HLF function ⁶ ⁷ ⁸. Thus, IPAS seems toinhibit more down-stream steps in signal transduction mediated byhypoxia-inducible transcription factors.

Example 5 IPAS Mediates Down-Regulation of Hypoxia-Inducible GeneExpression-IPAS Impairs Interaction Between HIF-1 α and the HRE

To further investigate the role of IPAS in regulation of HIF-mediatedsignaling pathways in hypoxic cells, we generated cells stablyoverexpressing IPAS by stable transfection of mouse hepatoma Hepa1c1c7cells. Expression of IPAS mRNA in the stably transfected cells wasconfirmed by Northern blot analysis, whereas the parental Hepa1c1c7cells did not show any detectable endogenous IPAS expression (FIG. 3 a).Wild type Hepa1c1c7 cells cultured under hypoxic conditions showedmarkedly increased expression of mRNAs encoding phosphoglycerate kinase1(PGK1) and vascular endothelial growth factor (VEGF) (FIG. 3 a), both ofwhich have been demonstrated to be induced under hypoxic conditions in avariety of cell lines ⁹ ¹⁰. In response to hypoxia, Hepa IPAS cellsshowed decreased levels of induction of these genes (45% and 48%reduction in PGK1 and VEGF activation, respectively; FIG. 3 a).IPAS-mediated down-regulation of hypoxia-inducible gene expressionseemed to be at the transcriptional level, since activation of atransiently transfected HRE-driven reporter gene by hypoxia wassignificantly lower in Hepa IPAS cells than in wild type cells (FIG. 3b). Reporter gene activation was even suppressed in Hepa IPAS cellsfollowing transient overexpression of HIF-1α, indicating that IPASimpairs productive interaction between HIF-1α and the HRE.

Example 6 IPAS Impairs DNA Binding Activity of the HIF-1 α/Arnt Complex

Mobility shift assay was carried out as described by Gradin et al.¹². Instrong support of the results obtained in Example 5, HRE-specific DNAbinding activity by the HIF-1α/Arnt heterodimeric complex was lower innuclear extract from either normoxic or hypoxic Hepa IPAS cells thancorresponding nuclear extracts from wild-type cells (FIG. 3 c).

Example 7 Negative Regulation by IPAS is Specific to HIF-MediatedSignaling Pathways.

It was examined whether negative regulation by IPAS is specific toHIF-mediated signaling pathways. The aryl hydrocarbon receptor (AhR),which mediates gene regulation in response to xenobiotic chemicals, isalso a member of the bHLH/PAS transcription factor family and shares thedimerization partner factor Arnt with HIF-1 α¹¹. Incubation of thewild-type Hepa 1c1c7 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)massively induced mRNA expression of the AhR target gene cytochromeP-4501A1(CYP1A1). In contrast to hypoxia-inducible gene expression,TCDD-induced expression of CYP1A1mRNA was unperturbed in Hepa IPAS cellswhich showed an induction response similar to that observed in wild typecells (FIG. 3 d). Consistent with these data, very similar levels ofactivation of a xenobiotic response element (XRE)-driven reporter geneby the ligand-stimulated AhR/Arnt heterodimeric complex ¹² was observedboth in the wild type and the IPAS overexpressing Hepa cells (FIG. 3 e).Taken together, IPAS seems to preferentially target HIF-1 α to act as adominant negative regulator of hypoxia-inducible gene expression.

Example 8 The inhibitory action of IPAS is mediated by directinteraction with HIF-1α

It was tested whether the inhibitory action of IPAS is mediated bydirect interaction with HIF-1α or Arnt. Radiolabeled, in vitrotranslated HIF-1α or Arnt were incubated with glutathione S-transferase-(GST-) IPAS fusion proteins and analyzed by immunoprecipitation assaysusing anti-GST antibodies (Amersham Pharmacia Biotech). GST-IPAS wascoprecipitated with HIF-1 α but not with Arnt, demonstrating specificphysical interaction between IPAS and HIF-1α (FIG. 4 a).

Example 9 The N-Terminal Part of HIF-1α is Responsible for the PhysicalInteraction With IPAS

To identify the domain of HIF-1α essential for interaction with IPAS, weincubated various fragments of HIF-1α (see Kallio et al.; ref. 18) fusedto the GAL4 minimal DNA binding domain and radiolabeled IPAS generatedby in vitro translation, and immunoprecipitated this material by antiGAL-4 antibodies (Upstate Biotechnology). GALA-4-HIF-1α/1-826 (fulllength), /1-330, and/1-652 clearly coprecipitated IPAS whereasGAL4-HIF-1α/526-826 and GAL4 DBD did not. Together, N-terminal structureof HIF-1 α mainly composed of bHLH/PAS motif is responsible for thephysical association with IPAS (FIG. 4 b). In support of theseobservations, mammalian two-hybrid assay employing GAL4-HIF-1α/1-330 andVP16-IPAS demonstrated interaction between IPAS and N-terminal part ofHIF-1α in the cells (FIG. 4 c). On the other hand, in analogy to theresults from pull down assay, GAL-4-IPAS and VP16-Arnt failed to showany interaction (FIG. 4 d).

Example 10 IPAS Inhibits DNA Binding Activity of HIF-1 α/Arnt Complex

To elucidate the function of IPAS/HIF-1α complex, ElectrophoreticMobility Shift Assay using HRE oligonucleotide probe and in vitrotranslated proteins was performed. IPAS/HIF-1α heterodimer, as well asHIF-1α or IPAS by itself, was abortive in binding to HRE. ThusIPAS/HIF-1α complex seemed to be inactive in mediating expression of thegenes under control of HRE. Moreover, DNA binding activity ofHIF-1α/Arnt complex was inhibited by the copresence of IPAS but not bythe control translation product (FIG. 4 e), indicating that theIPAS/HIF-1α complex might functionally dominate over HIF-1α/Arnt DNAbinding complex.

Example 11 Introduction of IPAS Antisense into Cornea Cells StimulatesExpression and Hypoxia Inducibility of the VEGF Gene

What is the significance of dominant negative function of IPAS inhypoxia signaling and its massive expression in, for example, corneaepithelium? A hallmark of normal cornea is a total avascularity andmaintenance of transparency is essential to corneal function. By anovernight eye closure, corneal environment can be enough hypoxic tostimulate hypoxia-inducible gene expression ¹³ ¹⁴, however,neovascularization in cornea is usually prevented although underlyingmechanisms are unknown.

Given the fact IPAS down regulates hypoxia-responsive VEGF expression,we tried to elucidate the effect of IPAS on hypoxia-inducible VEGFexpression in cornea. For this purpose, a primary culture of the corneaepithelium cells was transfected with antisense IPAS expression plasmid(or control empty vector) to manipulate IPAS level and incubated with orwithout hypoxic stimulation for 24 h, thereafter VEGF mRNA expressionwas monitored in comparison with hepatoma cell lines by NorthernBlotting. Hepatoma cell lines showed high level induction of VEGF byhypoxic treatment as previously shown ¹⁰ ¹² (FIG. 5). In sharp contrast,cornea cells transfected with control vector demonstrated low basallevel and modest induction of VEGF expression by hypoxia, which mightrepresent the mechanism for a low profile of corneal angiogenesis.Strikingly, introduction of IPAS antisense into the cornea cellsrecovered both basal expression and hypoxia inducibility of VEGF gene(FIG. 5), indicating that IPAS may have an important role in silencingangiogenic VEGF expression in cornea especially in hypoxic conditions.

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1. An isolated mammalian IPAS polypeptide comprising the amino acidsequence set forth as SEQ ID NO:3.